Analogues of lipopolysaccharide-binding protein (LBP)-derived peptides that efficiently neutralize lipopolysaccharides (LPS)

ABSTRACT

The present invention is for a pharmaceutical composition comprising an effective amount of an lipopolysaccharide (LPS) binding and -neutralizing peptide. The LPS binding and neutralizing peptides of the present invention are analogues of peptides from a lipopolysaccharide binding protein region whose primary sequence have been substituted at particular amino acid sites to obtain an effective binding to and neutralization of LPS.

[0001] The present invention relates generally to analogues of peptidesfrom a LBP region whose primary sequence have been substituted atparticular amino acid (a.a.) sites to obtain an effective binding to andneutralization of LPS; and specifically to the use of the peptides, andtheir derivatives, to prevent and treat sepsis and otherendotoxin-related disorders.

[0002] Systemic inflammatory responses can be triggered by bothinfectious and not infectious disorders, such as severe trauma andpancreatitis. Sepsis include those manifestations related to thesystemic response to infection, like tachycardia, tachypnea, chills,initial irregularly remittent fever followed by persistent fever, andleukocytosis, and those related to the organs dysfunction, such ascardiovascular, respiratory, renal, hepatic and hematologicalabnormalities. Sepsis is considered severe when it is associated withsigns of hypoperfusion, like lactic acidosis, oliguria and alteredmental status, with hypotension leading to shock, or with disseminatedintravascular coagulation, adult respiratory distress syndrome andmultiple organ failure.

[0003] Toxins produced or released by diverse microorganisms initiatesthe sepsis pathogenic cascade. Although septic shock is often onlyassociated with Gram-negative bacteremia, Gram-positive bacteria, fungi,viruses, protozoa, spirochetes, rickettsiae, and inclusive plans andvenoms can produce septic shock syndromes. E.coil is the most commonlyisolated Gram-negative pathogen in sepsis, followed byKlebsiella-Enterobacter, and other bacteria such as Pseudomonas, Proteusand Serratia. Also Neisseria meningitidis bacteremia is a frequent causeof septic shock.

[0004] The process begins with the colonization by microorganisms of atissue nidus. Then the organisms may invade bloodstream directly(bacteremia) or may proliferate locally and release toxic substancesinto the bloodstream (toxemia). Among these released substances,endotoxin, an structural component of gram-negative bacteria outermembrane is commonly associated with sepsis.

[0005] Endotoxin or lipopolysaccharide (LPS) is an ubiquitous componentin the external leaf of the outer membrane of all Gram-negativebacteria. LPS biological and pharmacological activities are quitesimilar regardless the particular microorganisms they were derived of,or the specific strain pathogenicity Structural differences are observedin endotoxin derived from different gram-negative bacterial strains. Theoutermost part of the endotoxin molecule consists of a series ofoligosaccharides that are structurally and antigenically diverse.Internal to this oligosaccharides are the core saccharides which arestructurally rather similar in gram-negative bacteria. To the coreoligosaccharide is bound a lipid moiety, lipid A, highly conservedstructure that is responsible for most LPS toxicity and biologicalactivities.

[0006] LPS triggers both humoral and cell activation mechanisms thathave a primary pathogenic role in shock and organ failure. Varioushumoral pathways are activated by LPS including the complement,coagulation and kallikrein cascades, which are partially responsible forhaemodinamic changes observed in sepsis. Nevertheless, interactionsbetween LPS and cellular receptors in a variety of cell types play apivotal role in the biological and toxic effects of LPS. Particularlycells of the monocyte/macrophage lineage are involved in the hostprimary response to endotoxin. Other implicated cell types arepolymorphonuclear (PMN) leukocytes and endothelial cells Activation ofthese cell types by LPS is characterized by the rapid production andreleased of a series of products that constitute central endogenousmediators of sepsis, especially different cytokines such as TNF, IL-1and IL-6.

[0007] The intravascular activation of inflammatory systems involved inseptic shock, as the haemodinamic alterations, are mainly theconsequence of a dysregulation in the production of these cytokines. Oneof them, TNF, is now regarded as a central mediator of thepathophysiological changes associated with LPS release. Thereforeexperimental approaches that inhibits TNF release induced by LPS areattractive as potential procedures to reduce sepsis morbidity andmortality.

[0008] LPS interacts with cellular receptors that are linked to signalpathways mediating cellular activation. CD14 is a membraneglycerolphosphorylinositol-anchored protein (mCD14) that is currentlyconsidered the major cellular receptor for LPS in myeloid cells¹.Another protein, LBP, promotes interaction between LPS and mCD14 to forma high affinity complex. LBP enhances the binding of LPS to the membraneform of CD14, forming a ternary complex LBP: LPS: CD14. A soluble formof CD14 (sCD14) is also present in serum and it forms complexes with LPSthat activates LPS-responsive cells lacking mCD14 such as endothelial,smooth muscle and epithelial cells². LBP plays a catalytic role in theformation of LPS:sCD14 complexes for binding to non-mCD14 bearing cells.Therefore LBP plays a critical function in LPS-mediated cell activationevents observed in sepsis; molecules that have the ability to compete orinhibit LBP enhancing effects in LPS biologic functions, couldcontribute definitively to ameliorate symptoms of sepsis.

[0009] During the last decade various agents that neutralize LPS effectshave been described, and some of them are currently under preclinical orclinical development. Mortality induced by endotoxin administration wasreduced in experimental animals pre-treated with anti-LPS antibodies³.Inasmuch as the N-terminal fragment of bactericidal/permeabilityincreasing protein (BPI) keeps holoprotein's antiendotoxic andbactericial properties⁴, this fragment have been used in preclinical,and recently in clinical trials to asses its effectiveness in thetreatment of LPS-associated disorders such as meningococcemia,hemorrhagic trauma and severe intra-abdominal infections.

[0010] BPI-derived peptides and their analogues have been evaluated asendotoxin antagonists⁵⁻⁸. Among other peptide agents that bind to andneutralize LPS are those derived from Limulus anti-LPS factor (LALF)⁹and the synthetic antiendotoxin peptides mimicking polymyxin Bstructure.

[0011] The N-terminal fragment of LBP¹⁰, as well as other shorterpeptides, including 17-45, 65-108 and 142-169 regions, or segmentsthereof^(6,11-14), have been described as LPS binding regions and theirneutralizing ability over LPS toxic effects have been demonstrated.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to analogues of peptide sequencesderived from a LBP region, which result from substitutions within theprimary structure of the native protein, and have an improved ability tobind and neutralize LPS biological effects. The above mentioned a.a.substitutions render peptides with advantageous properties when comparewith previously described overlapping sequences^(6,10-14).

[0013] To understand the molecular basis of LPS-protein interaction thea.a. sequence of LBP and BPI, proteins known to bind specifically LPS,were analyzed. The aim of the study was to detect features in theprimary structure of these proteins that could be correlated with theability to interact with LPS. The analysis included predictions ofsecondary structure and accessibility, sequence similarity searches,inspection of conserved residues and analysis of the distribution ofcharged residues along the sequence. Inspection of the alignment revealsthat some linear clusters of basic residues are present in LBP and BPI.These moieties could favorably interact with acidic groups of the highlyanionic LPS (Lipid A). The basic character of these regions was specificof BPI and LBP; corresponding a.a. in other proteins from the samefamily are mainly neutral, with some aromatic residues. This fact couldreflect differences between ligands: phospholipid transfer protein(PLTP) and cholesteryl ester transfer protein (CETP) bind specificallyphospholipids and lipoproteins, respectively.

[0014] The analysis suggested that a potential LPS-binding site in LBPis located between residues 89 and 96 of the mature protein, consideringa major cluster of basic residues in this region¹⁴. Other authors alsoproposed this, or overlapping sequences, as potential LPS-binding sitesin LBP^(6,11-13,15).

[0015] To corroborate this hypothesis, synthetic peptides correspondingto this region, including or not selected substitutions in specificresidues were designed, synthesized and tested¹⁴.

[0016] This invention provides peptides whose sequences result fromsingle or multiple a.a substitutions at selected sites of the series,regarding the native sequence, which optimize the neutralizing capacityof the analogues.

[0017] In a first embodiment the invention relates to peptidescharacterized by their ability to antagonize the LBP:LPS interaction andto inhibit the biological effects triggered by LPS. The peptides of thisinvention present essential a.a. at positions +1, +5, +6, +7, +9, +10,+11, +12 and +13, and other preferred a.a. in other positions within thedescribed sequences, all necessary for the optimal display of theLPS-neutralizing properties of the peptides. Herein “essential” a.a. aredefined as those indispensable at said positions for displaying improvedLPS-neutralizing properties.

[0018] In a preferred embodiment the present invention relates topeptides that bind to and efficaciously neutralize LPS, whose a.a.sequence is derived from the 86-99 a.a. region of the LBP mature protein(SEQ. ID NO.1) but with selected substitutions at particular siteswithin this domain.

[0019] Preferred peptides of the present invention are those with thea.a. sequence X-1-2-3-4-5-6-7-8-9-10-11-12-13-14-Y, wherein:

[0020] X is a linear chain from zero to four amino acids.

[0021] (1) is one of the a.a. alanine, threonine, glutamine, asparagineor serine, and if and only if at least one of the a.a. at positions +5,+9, +10, +11 or +13 has been replaced (from the native sequence)according to what is herein described, then (1) could also be arginineor lysine.

[0022] (2) is one of the a.a. alanine, valine, isoleucine, leucine,phenylalanine, methionine, tryptophan or tyrosine.

[0023] (3) is one of the a.a. glutamine, asparagine, serine orthreonine.

[0024] (4) is one of the a.a. glycine, alanine, valine, isoleucine,leucine, phenylalanine, methionine, tryptophan or tyrosine.

[0025] (5) is one of the a.a. alanine, threonine, glutamine, asparagineor serine, and if and only if at least one of the a.a. at positions +1,+9, +10, +11 or +13 has been replaced according to what is hereindescribed, then (5) could also be arginine or lysine.

[0026] (6) is one of the a.a. tryptophan or phenylalanine.

[0027] (7) is one of the a.a. lysine or arginine.

[0028] (8) is one of the a.a. alanine, valine, isoleucine, leucine,phenylalanine or tyrosine.

[0029] (9) is one of the a.a alanine, threonine, glutamine, asparagineor serine, and if and only if at least one of the a.a at positions +1,+5, +10, +11 or +13 has been replaced according to what is hereindescribed, then (9) could also be arginine or lysine.

[0030] (10) is one of the a.a. alanine, valine, isoleucine, leucine,phenylalanine, methionine, tryptophan or tyrosine, and if and only if atleast one of the a.a. at positions +1, +5, +9, +11 6 +13 has beenreplaced according to what is herein described, then (10) could also belysine or arginine.

[0031] (11) is one of the a.a. alanine or valine; and if and only if atleast one of the a.a. at positions +1, +5, +9, +10, 6 +13 has beenreplaced according to what is herein described, then (11) could also beserine; and if and only if the a.a. at position +10 has been replacedaccording to what is herein described, then (11) could also bethreonine, glutamine, asparagine, lysine or arginine.

[0032] (12) is one of the a.a. phenylalanine, tryptophan or tyrosine.

[0033] (13) is one of the a.a. alanine, threonine, glutamine, asparagineor serine; and if and only if at least one of the a.a. at positions +1,+5, +9, +10 6 +11 has been replaced according to what is hereindescribed, then (13) could also be phenylalanine, arginine or lysine;and if and only if the a.a at position +14 is lysine or arginine, then(13) could also be glycine.

[0034] (14) is one of the a.a. lysine, arginine or alanine, and if andonly if the a.a. at position +13 has been replaced according to what isherein described, then (14) could also be valine, isoleucine, leucine,phenylalanine, methionine, tryptophan or tyrosine.

[0035] Y is a linear chain from zero to four or amino acids.

[0036] The a.a. residues in the previously described preferred peptidescould be D- or L-amino acids.

[0037] Representative examples of specifically preferred peptides of thepresent invention include sequences ID no. 2 to 52.

[0038] Tests indicate that peptides having the above described sequencesshow advantageous properties when compared with peptides having othera.a. at the selected sites, or that are substituted at these positionswithout considering the definitions of the present invention.

[0039] Specifically, peptides with the above described sequences haveadvantageous properties respect to peptides with sequences correspondingto the native LBP protein, or to others including segments of themshorter than 8 a.a.

[0040] The functional superiority of the peptides of the presentinvention respect to previously described peptides¹⁰⁻¹⁴ is based inthese single substitutions, and their combinations. These previousreports described peptides derived from LBP with LPS-binding and-neutralizing properties, which could include partially or totally theherein selected sequence, but maintain the LBP native primary sequenceor do not involve the essential single or combined substitutionsdescribed in the present invention. The neutralizing potency of thepeptides described in previous studies, which do not include theessential substitutions as defined in this invention, is various timeslower than the potency of the peptides of the present invention, asshown in examples 1 and 3. In addition, peptides that are substituted,in regard to the LBP primary sequence, as defined in SEQ ID No. 53, 54and 55, or have other a.a different to those defined at the presentinvention for positions +6, +7 and +12 lack of relevant LPS-neutralizingcapacity, as shown in the examples 1 to 3.

[0041] Substitutions at the other positions (+1, +5, +9, +10, +11 y+13). as described in the present invention and how illustrate preferredpeptides (SEQ ID No. 2 to 52), increased considerably and unexpectedlythe peptide ability to block LPS:LBP interaction, and enhanced theinhibitory effect upon LPS-mediated activation of inflammatory cells.

[0042] This unexpected quality exhibited by the peptide analogues of theinvention differs from the effect of some of these same substitutionswithin the holoprotein, as described by others¹⁶. This fact remarks thedistinction between the interaction of the peptides of the invention andLPS, and that of LBP, or even its functional N-terminal fragment.

[0043] This invention also relates to peptide analogues whit thedescribed sequences which are constrain to adopt a cyclic conformationby means of a disulfide bond formed between two cysteine residues addedto their N- and C-terminus respectively, or through an amide bond formedbetween the side chains of constituting amino acids.

[0044] It is further contemplated in this invention that those skill inthe art are able to replace particular amino acid residues by nonnaturalhomologous amino acids maintaining the LPS-neutralizing properties ofthe whole molecule, as well as to change the main chain backbone bybackbone-mimetic organic compounds.

[0045] In another embodiment this invention relates to largerpolypeptides bearing the above described preferred sequences at their N-or C-terminus in such a way that maintains the ability to bind andneutralize LPS and confers this ability to the hybrid polypeptide. Apreferred hybrid polypeptide comprises a fusion of any of the preferredpeptides and light or heavy chain regions of immunoglobulins (Ig),including the insertion of the peptide sequences of the invention withinthe framework of the Ig molecule

[0046] This invention also relates to scaffold proteins thatappropriately exposed one of the preferred peptide sequences in such away that maintains or enhances their ability to bind and neutralize LPSand confers this ability to the hybrid polypeptide. The term “scaffoldproteins” as herein used refers to hybrid polypeptides that includewithin their polypeptide chain one or more of the selected sequences insuch a way that the inserted segment forms an exposed loop in thestructure of the fused protein or polypeptide.

[0047] Also this invention relates to two or more repeats of one of thepreferred polypeptide sequences in a linear polypeptide chain, or thecombination of two or more of them, in such a way that these sequencesare connected by linkers, and the novel polypeptide have the ability tobind and efficiently neutralize LPS. Preferred linkers are those havingbetween 12 and 25 amino acid residues and are rich in the glycine,alanine, proline or serine residues. Likewise the present inventionapplies to arrangements of three or more copies of the preferred peptidesequences linked by their C-terminus to a lysine core, formingstructures that have the ability to bind and efficaciously neutralizeLPS. Other arrangements of preferred sequences could result from thecombination of the aforementioned cyclic peptides.

[0048] Synthetic peptides having the described preferred sequences aresmall molecules with broader utility than larger polypeptides.Particularly, the peptides of the present invention will have someadvantages over larger polypeptides concerning immunogenicity andspectrum of LPS-neutralizing activity. In vivo half-life and otherpharmacological parameters of the peptides could be improved with hybridand scaffold polypeptides and proteins bearing the preferred sequences.

[0049] All the peptides encompassed by the present invention can beprepared using standard procedures of peptide synthesis, including forexample the solid-phase synthetic technique describe by Merrifield¹⁷, aswell as other apparent to anyone skilled in the art.

[0050] In a further preferred embodiment this invention providespharmaceutical compositions comprising pharmaceutically appropriateddiluents, carriers or adjuvants, and effective quantities of one of moreof the peptides, or hybrid or scaffold proteins containing theirsequences. The term “effective quantity” as herein used refers to theamount of the peptides, or hybrid or scaffold proteins, that issufficient to ameliorate symptoms associated with systemic responses toLPS.

[0051] The novel pharmaceutical compositions can be useful for methodsto treat various disorders associated with the release of LPS, speciallythe infection with Gram-negative bacteria and its sequelae: endotoxemiaand shock, Systemic Inflammatory Response Syndrome (SIRS), CompensatoryAnti-inflammatory Response Syndrome (CARS), disseminated intravascularcoagulation, Adult Respiratory Distress Syndrome and Multiple OrganDysfunction Syndrome (MODS). The therapeutic method is provided toameliorate one or more symptoms of patients suffering or at risk fordeveloping disorders caused by diverse insults such as infection,trauma, burns and pancreatitis. Patients who also may require such atreatment include those afflicted from inflammatory bowel diseases andobstructive jaundice or other disorders where gastrointestinalpermeability is impaired and bacterial translocation or endotoxinleakage occur.

EXAMPLES Example 1

[0052] This example describes the capacity of preferred peptides of theinvention to block the interaction between LBP and E.coli LPS. An ELISAwas used to determine the binding of biotinylated-LPS tosurface-captured human LBP, in the presence or absence of fixedquantities of the selected peptides. LPS was biotinylated according tostandard procedures. Human LBP (hLBP) was captured by using a specificmonoclonal antibody, purified by affinity chromatography. Mixtures ofLPS and each peptide were incubated during 2 h at room temperature, andthen 100 μL were added to hLBP-containing wells. The binding ofbiotinylated-LPS to hLBP was detected with horseradishperoxidase-conjugated streptavidin. The assay was developed by adding achromogenic substrate. FIG. 1 shows the results of this assay whenbiotinylated-LPS was incubated with peptides LBP₈₆₋₉₉ (LBP), LBP_(A86)(SEQ ID No.2), LBP_(A90) (SEQ ID No.3) and LBP_(A94) (SEQ ID No.4). FIG.2 shows results for peptides LBP_(A95) (SEQ ID No.5), LBP_(A96) (SEQ IDNo.6) and LBP_(A98) (SEQ ID No.7), and FIG. 3 represents the results forpeptides LBP_(A91) (SEQ ID No.55), LBP_(A92) (SEQ ID No.53) andLBP_(A97) (SEQ ID No.54). Each experiment recorded the interaction ofhLBP and biotinylated-LPS in absence of peptides, in presence ofLBP₈₆₋₉₉, and in the example presented in FIG. 2 the effect of anon-related cationic peptide (C5,3).

[0053] The interaction between hLBP and E.coli LPS was notably impairedby peptides of this invention, as represented by LBP_(A86), LBP_(A90),LBP_(A94), LBP_(A95), LBP_(A96) and LBP_(A98), and it was not affectedby peptides LBP_(A91), LBP_(A92) and LBP_(A97).

[0054] These results demonstrate that peptides with sequences defined asspecially preferred in this invention have higher blocking capacity ofLBP:LPS interaction than the LBP₈₆₋₉₉, that has the native sequence ofthis region in human LBP. This example confirms that the a.a.substitutions described in the present invention for peptide sequencesderived from this particular region of hLBP are essential to obtainpeptide analogues that efficaciously block the interaction between hLBPand LPS. Likewise, this example demonstrates that replacements atparticular sites of the series, by different a.a. to those described inthis invention, reduce or abrogate the LPS-neutralizing activity ofpeptides derived from the mentioned hLBP region.

Example 2

[0055] In order to determine if the peptides of the present inventionwere able to neutralize LPS-mediated responses, their ability to reducethe release of TNF by LPS-activated human peripheral blood cells wasestimated. This assay evaluates the release of TNF by LPS-induced humanperipheral blood mononuclear cells (PBMC), using concentrations of LPScommonly found in septic patients. LPS was incubated with fixedconcentrations of each peptide during 2 h at 37° C. and the mixtureswere then added to PBMC. The culture medium was supplemented with humanLBP (200 ng/mL) and plates were incubated at 37° C. in a 5% CO₂atmosphere. TNFA was measured in culture supernatants after 18 h using ahuman TNF-α specific ELISA. FIG. 4 represents the average results fromthree different experiments. Reduced levels of cytokine release wereobserved in cultures containing E.coli 0111:B4 LPS and one of thefollowing peptides: LBP₈₆₋₉₉, LBP_(A94), LBP_(A95) and LBP_(A96). Thelast three peptides exhibited higher inhibition than LBP₈₆₋₉₉ (LBP).Similar results were observed when other concentrations of LPS (2 or 10ng/mL) and hLBP (20 or 100 ng/mL) were used. The non-related, cationicpeptide B6,1 did not modify, as expected, the release of TNF in thisassay. On the other hand the LBP_(A91) peptide, which has tryptophanresidue at position (6) replaced with alanine, did not inhibitLPS-stimulated TNF production.

[0056] These results demonstrate that the peptides derived from thisregion of hLBP should have the specified sequences of this invention fordisplaying vigorous inhibitory activity upon LPS-mediated activation ofhuman mononuclear cells. Likewise, the results suggest the usefulness ofthe peptides of the present invention for developing prophylactic andtherapeutic methods for sepsis, systemic inflammatory response syndromeand other related disorders. The potency of the preferred peptides ofthis invention is properly demonstrated in this example, where endotoxinconcentrations commonly found in endotoxemic patients were used.

Example 3

[0057] This invention provides peptides with advantageous functions overother LBP-derived peptides that have the native sequence or differenta.a. replacements to those herein described at positions +1, +5, +6, +7,+9, +10, +11, +12 and +13. Among these others are some peptidespreviously described by their ability to reduce LPS biologicaleffects¹¹⁻¹⁴. In order to demonstrate the advantage of the preferredpeptides of this invention over these previously described peptides,their antagonist activity upon LPS-induced responses was compared; theeffect of these different peptides on the LPS-induced IL-6 production incultures of human PBMC is illustrated in FIG. 5. The experimentalprocedure was similar to that one described in EXAMPLE 2. IL-6 wasmeasured in culture supernatants after 18 h using a human IL-6 specificELISA. In the represented experiment, the following peptides wereevaluated: LBP₈₆₋₉₉, LBP-H (LBP₈₆₋₁₀₁, SEQ ID No.56)¹³, LBP_(A94),LBP_(A95) and LBP_(A98). IL-6 production was inhibited more than 35%only by LBP_(A94), LBP_(A95) and LBP_(A96), which have essential a.a.,as defined in the present invention, at positions +9, +10, and +13respectively. LBP_(A95) reduced the LPS-triggered response more than70%, at every hLBP concentrations tested. This example remarks thecapacity of the peptides of this invention to reduce the production ofpro-inflammatory cytokines by LPS-stimulated cells.

Example 4

[0058] An endotoxin shock animal model was used to determine If thepeptides of this invention were able to block complex physiologicalresponses triggered by LPS. In this model mice were sensitized withActinomycin D to increase LPS-mediated toxic responses. With thispurpose, Actinomycin D (7.5 μg) was administered i.p. to 6 to 8weeks-old female mice. Simultaneously each mice received LPS, andsurvival was evaluated every 24 h during 120 h.

[0059] The effect of peptides of the present invention on mice survivalwas assessed by administering E.coli LPS (1 μg/mouse in saline vehicle)or LPS:peptide mixtures mixtures (previously incubated during 1 h at 37°C.) to BALB/c sensitized mice. FIG. 6 represents the results of arepresentative experiment where the following peptides were eachadministered at equimolar doses to groups of 20 mice: LBP₈₉₋₉₉,LBP_(A94), LBP_(A95), LBP_(A98) or B6,1 (cationic, non-related peptide).Mice survival was only significantly increased by LBP_(A94), LBP_(A95)or LBP_(A96) (*p<0.05 vs. vehicle or B6,1). The peptide LBP₈₆₋₉₉increase survival only marginally. This example demonstrates the higherefficacy of preferred peptides of this invention in protecting mice fromLPS lethal inocula. The aforementioned in vivo neutralizing property ofthe peptides of the invention is relevant for their application inprophylactic or therapeutic methods for sepsis and other associateddisorders.

[0060] Thus the above-mentioned results indicate that peptides of thisinvention retain their LPS-blocking properties when tested underpatho-physiological conditions, demonstrating their pharmacologicalpotency.

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Analogues of Lipopolysaccharide-binding Protein (LBP)-derivedPeptides that Efficiently Neutralize Lipopolysaccharides (LPS)

[0062]FIG. 1 shows the inhibition of the interaction between E.coli LPSand hLBP by preferred peptides of the present invention (LBP_(A86),LBP_(A90) and LBP_(A94)). Human LBP was captured to the plates using aspecific monoclonal antibody. Binding of biotinylated-LPS was detectedwith an streptavidin-horseradish peroxidase conjugate. The extent towhich the peptide LBP₈₆₋₉₉ (LBP) inhibits this interaction is alsoshown.

[0063]FIG. 2 shows the inhibition of the interaction between E.coli LPSand hLBP by preferred peptides of the present invention (LBP_(A95),LBP_(A96) and LBP_(A98)). The experimental conditions were similar tothose described in FIG. 1. The effect of a non-related cationic peptide,C5,3 is also included.

[0064]FIG. 3 shows the effect on the interaction of hLBP and E.coli LPSof peptides that have distinct residues at positions +6, +7 or +12 tothose described in this invention (LBP_(A91), LBP_(A92) and LBP_(A97)).The experimental conditions were similar to those described in FIG. 1.

[0065]FIG. 4 shows the inhibition by peptides of the present invention(LBP_(A94), LBP_(A95) and LBP_(A98)) of the LPS-mediated release of TNFby human PBMC. The effects on this assay of LBP₈₆₋₉₉ (LBP), LBP_(A91),B6,1 and polymyxin B (PMB) are also shown.

[0066]FIG. 5 shows the inhibition by peptides of the present invention(LBP_(A94), LBP_(A95) and LBP_(A96)) of the LPS-mediated release of IL-6by human PBMC. Results are expressed as % inhibition of IL-6 releasecompared with the cytokine production in the absence of peptides. Theeffects on this assay of LBP₈₆₋₉₉ (LBP), LBP-H and B6,1 are also shown.

[0067]FIG. 6 shows survival data of groups of 20 mice each challengedwith E.coli LPS i.p. and simultaneously treated with equimolar amountsof different peptides of the present invention. Survival in groups ofBALB/c mice treated with LBP₈₆₋₉₉ (LBP) and B6,1 is also shown. Micesurvival was recorded each 24 h during 120 h.

1 56 1 14 PRT Artificial Sequence Description of Artificial Sequencepeptide 1 Arg Val Gln Gly Arg Trp Lys Val Arg Lys Ser Phe Phe Lys 1 5 102 14 PRT Artificial Sequence Description of Artificial Sequence peptide2 Ala Val Gln Gly Arg Trp Lys Val Arg Lys Ser Phe Phe Lys 1 5 10 3 14PRT Artificial Sequence Description of Artificial Sequence peptide 3 ArgVal Gln Gly Ala Trp Lys Val Arg Lys Ser Phe Phe Lys 1 5 10 4 14 PRTArtificial Sequence Description of Artificial Sequence peptide 4 Arg ValGln Gly Arg Trp Lys Val Ala Lys Ser Phe Phe Lys 1 5 10 5 14 PRTArtificial Sequence Description of Artificial Sequence peptide 5 Arg ValGln Gly Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 6 14 PRTArtificial Sequence Description of Artificial Sequence peptide 6 Arg ValGln Gly Arg Trp Lys Val Arg Lys Ala Phe Phe Lys 1 5 10 7 14 PRTArtificial Sequence Description of Artificial Sequence peptide 7 Arg ValGln Gly Arg Trp Lys Val Arg Lys Ser Phe Ala Lys 1 5 10 8 14 PRTArtificial Sequence Description of Artificial Sequence peptide 8 Ala ValGln Gly Arg Trp Lys Val Arg Lys Ser Phe Ala Lys 1 5 10 9 14 PRTArtificial Sequence Description of Artificial Sequence peptide 9 Ala ValGln Gly Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 10 14 PRTArtificial Sequence Description of Artificial Sequence peptide 10 AlaVal Gln Gly Ala Trp Lys Val Arg Lys Ser Phe Phe Lys 1 5 10 11 14 PRTArtificial Sequence Description of Artificial Sequence peptide 11 AlaVal Gln Gly Arg Trp Lys Val Ala Lys Ser Phe Phe Lys 1 5 10 12 14 PRTArtificial Sequence Description of Artificial Sequence peptide 12 AlaVal Gln Gly Arg Trp Lys Val Arg Lys Ala Phe Phe Lys 1 5 10 13 14 PRTArtificial Sequence Description of Artificial Sequence peptide 13 ArgVal Gln Gly Ala Trp Lys Val Ala Lys Ser Phe Phe Lys 1 5 10 14 14 PRTArtificial Sequence Description of Artificial Sequence peptide 14 ArgVal Gln Gly Ala Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 15 14 PRTArtificial Sequence Description of Artificial Sequence peptide 15 ArgVal Gln Gly Ala Trp Lys Val Arg Lys Ala Phe Phe Lys 1 5 10 16 14 PRTArtificial Sequence Description of Artificial Sequence peptide 16 ArgVal Gln Gly Arg Trp Lys Val Ala Lys Ala Phe Phe Lys 1 5 10 17 14 PRTArtificial Sequence Description of Artificial Sequence peptide 17 ArgVal Gln Gly Arg Trp Lys Val Ala Lys Ser Phe Ala Lys 1 5 10 18 14 PRTArtificial Sequence Description of Artificial Sequence peptide 18 ArgVal Gln Gly Arg Trp Lys Val Arg Lys Ala Phe Ala Lys 1 5 10 19 14 PRTArtificial Sequence Description of Artificial Sequence peptide 19 ArgVal Gln Gly Arg Trp Lys Val Arg Ala Ser Phe Ala Lys 1 5 10 20 14 PRTArtificial Sequence Description of Artificial Sequence peptide 20 ArgPhe Gln Gly Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 21 14 PRTArtificial Sequence Description of Artificial Sequence peptide 21 ArgVal Asn Gly Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 22 14 PRTArtificial Sequence Description of Artificial Sequence peptide 22 ArgVal Gln Met Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 23 14 PRTArtificial Sequence Description of Artificial Sequence peptide 23 ArgVal Gln Phe Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 24 14 PRTArtificial Sequence Description of Artificial Sequence peptide 24 ArgVal Gln Gly Arg Trp Lys Phe Arg Ala Ser Phe Phe Lys 1 5 10 25 14 PRTArtificial Sequence Description of Artificial Sequence peptide 25 ArgVal Gln Gly Arg Trp Lys Val Arg Ala Gln Phe Phe Lys 1 5 10 26 14 PRTArtificial Sequence Description of Artificial Sequence peptide 26 ArgVal Gln Gly Arg Trp Lys Val Arg Ala Ser Trp Phe Lys 1 5 10 27 14 PRTArtificial Sequence Description of Artificial Sequence peptide 27 ArgVal Gln Gly Arg Trp Lys Val Arg Ala Ser Phe Phe Ala 1 5 10 28 14 PRTArtificial Sequence Description of Artificial Sequence peptide 28 ArgVal Gln Gly Arg Trp Lys Val Arg Ala Ser Phe Gln Val 1 5 10 29 14 PRTArtificial Sequence Description of Artificial Sequence peptide 29 AlaVal Gln Gly Arg Trp Lys Val Arg Ala Ser Phe Thr Val 1 5 10 30 14 PRTArtificial Sequence Description of Artificial Sequence peptide 30 ArgVal Gln Gly Arg Trp Lys Val Arg Val Ser Phe Phe Lys 1 5 10 31 14 PRTArtificial Sequence Description of Artificial Sequence peptide 31 AlaVal Gln Gly Arg Trp Lys Val Arg Val Ser Phe Phe Lys 1 5 10 32 14 PRTArtificial Sequence Description of Artificial Sequence peptide 32 ArgVal Gln Gly Arg Trp Lys Val Arg Val Ser Phe Ala Lys 1 5 10 33 14 PRTArtificial Sequence Description of Artificial Sequence peptide 33 ArgVal Gln Gly Arg Trp Lys Val Arg Val Ser Phe Gln Val 1 5 10 34 14 PRTArtificial Sequence Description of Artificial Sequence peptide 34 ArgVal Gln Gly Arg Trp Lys Val Arg Val Thr Phe Phe Lys 1 5 10 35 14 PRTArtificial Sequence Description of Artificial Sequence peptide 35 ArgVal Gln Gly Arg Trp Arg Val Arg Val Lys Phe Thr Val 1 5 10 36 14 PRTArtificial Sequence Description of Artificial Sequence peptide 36 ArgVal Gln Gly Arg Trp Arg Val Arg Val Ala Phe Ala Lys 1 5 10 37 14 PRTArtificial Sequence Description of Artificial Sequence peptide 37 AlaVal Gln Gly Arg Trp Arg Val Arg Val Ser Phe Ala Lys 1 5 10 38 14 PRTArtificial Sequence Description of Artificial Sequence peptide 38 AlaVal Gln Gly Arg Trp Arg Val Arg Val Ser Phe Gln Val 1 5 10 39 14 PRTArtificial Sequence Description of Artificial Sequence peptide 39 ArgVal Ser Gly Arg Trp Arg Val Arg Val Ser Phe Gln Val 1 5 10 40 14 PRTArtificial Sequence Description of Artificial Sequence peptide 40 ArgVal Gln Gly Arg Trp Arg Val Arg Val Thr Phe Gln Val 1 5 10 41 14 PRTArtificial Sequence Description of Artificial Sequence peptide 41 ArgVal Gln Gly Arg Trp Arg Val Ala Lys Ser Phe Gln Val 1 5 10 42 14 PRTArtificial Sequence Description of Artificial Sequence peptide 42 AlaVal Gln Gly Arg Trp Arg Val Ala Lys Ser Phe Gly Lys 1 5 10 43 14 PRTArtificial Sequence Description of Artificial Sequence peptide 43 AlaVal Gln Gly Arg Trp Arg Val Ala Lys Ser Phe Gln Val 1 5 10 44 14 PRTArtificial Sequence Description of Artificial Sequence peptide 44 AlaVal Ser Gly Arg Trp Arg Val Ala Lys Ala Phe Gly Lys 1 5 10 45 14 PRTArtificial Sequence Description of Artificial Sequence peptide 45 ArgVal Gln Gly Ala Trp Lys Val Arg Ala Ser Phe Ala Lys 1 5 10 46 14 PRTArtificial Sequence Description of Artificial Sequence peptide 46 ArgVal Gln Gly Ala Trp Lys Val Arg Ala Ser Phe Gln Val 1 5 10 47 14 PRTArtificial Sequence Description of Artificial Sequence peptide 47 AlaVal Gln Gly Ala Trp Lys Val Arg Ala Ser Phe Ala Lys 1 5 10 48 14 PRTArtificial Sequence Description of Artificial Sequence peptide 48 AlaVal Gln Gly Ala Trp Lys Val Arg Ala Ser Phe Gln Val 1 5 10 49 18 PRTArtificial Sequence Description of Artificial Sequence peptide 49 ThrIle Arg Val Gln Gly Arg Trp Lys Val Arg Ala Ser Phe Phe Lys 1 5 10 15Leu Gln 50 18 PRT Artificial Sequence Description of Artificial Sequencepeptide 50 Thr Val Arg Val Gln Gly Ala Trp Lys Val Arg Ala Ser Phe PheLys 1 5 10 15 Leu Gln 51 18 PRT Artificial Sequence Description ofArtificial Sequence peptide 51 Thr Val Arg Val Gln Gly Arg Trp Lys ValArg Ala Ser Phe Ala Lys 1 5 10 15 Leu Gln 52 17 PRT Artificial SequenceDescription of Artificial Sequence peptide 52 Ser Val Arg Val Gln GlyArg Trp Lys Val Arg Ala Ser Phe Ala Val 1 5 10 15 Thr 53 14 PRTArtificial Sequence Description of Artificial Sequence example of apeptide substituted, in regard to the LBP primary sequence 53 Arg ValGln Gly Arg Trp Ala Val Arg Lys Ser Phe Phe Lys 1 5 10 54 14 PRTArtificial Sequence Description of Artificial Sequence example of apeptide substituted, in regard to the LBP primary sequence 54 Arg ValGln Gly Arg Trp Lys Val Arg Lys Ser Ala Phe Lys 1 5 10 55 14 PRTArtificial Sequence Description of Artificial Sequence example of apeptide substituted, in regard to the LBP primary sequence 55 Arg ValGln Gly Arg Ala Lys Val Arg Lys Ser Phe Phe Lys 1 5 10 56 14 PRTArtificial Sequence Description of Artificial Sequence peptide L 56 GlnGly Arg Trp Lys Val Arg Lys Ser Phe Phe Lys Leu Gln 1 5 10

1. An LPS-binding and -neutralizing peptide comprising the amino acid sequence X-1-2-3-4-5-6-7-8-9-10-11-12-13-14-Y, wherein: X is a linear chain from zero to four amino acids. (1) is one of the amino acids alanine, threonine, glutamine, asparagine or serine; and if and only if at least one of the a.a. at positions +5, +9, +10, +11 or +13 has been replaced (from the native LBP sequence) according to what is herein described, then (1) could also be arginine or lysine. (2) is one of the amino acids alanine, valine, isoleucine, leucine, phenylalanine, methionine, tryptophan or tyrosine (3) is one of the amino acids glutamine, asparagine, serine or threonine. (4) is one of the amino acids glycine, alanine, valine, isoleucine, leucine, phenylalanine, methionine, tryptophan or tyrosine. (5) is one of the amino acids alanine, threonine, glutamine, asparagine or serine; and if and only if at least one of the a.a. at positions +1, +9, +10, +11 or +13 has been replaced according to what is herein described, then (5) could also be arginine or lysine. (6) is one of the amino acids tryptophan or phenylalanine. (7) is one of the amino acids lysine or arginine. (8) is one of the amino acids alanine, valine, isoleucine, leucine, phenylalanine or tyrosine. (9) is one of the amino acids alanine, threonine, glutamine, asparagine or serine; and if and only if at least one of the a.a. at positions +1, +5, +10, +11 or +13 has been replaced according to what is herein described, then (9) could also be arginine or lysine. (10) is one of the amino acids alanine, valine, isoleucine, leucine, phenylalanine, methionine, tryptophan or tyrosine; and if and only if at least one of the a.a. at positions +1, +5, +9, +11 6 +13 has been replaced according to what is herein described, then (10) could also be lysine or arginine. (11) is one of the amino acids alanine or valine; and if and only if at least one of the a.a. at positions +1, +5, +9, +10, 6 +13 has been replaced according to what is herein described, then (11) could also be serine; and if and only if the a.a. at position +10 has been replaced according to what is herein described, then (11) could also be threonine, glutamine, asparagine, lysine or arginine. (12) is one of the amino acids phenylalanine, tryptophan or tyrosine. (13) is one of the amino acids alanine, threonine, glutamine, asparagine or serine; and if and only if at least one of the a.a. at positions +1, +5, +9, +10 6 +11 has been replaced according to what is herein described, then (13) could also be phenylalanine, arginine or lysine; and if and only if the a.a at position +14 is lysine or arginine, then (13) could also be glycine. (14) is one of the amino acids lysine, arginine or alanine, and if and only if the a.a. at position +13 has been replaced according to what is herein described, then (14) could also be valine, isoleucine, leucine, phenylalanine, methionine, tryptophan or tyrosine. Y is a linear chain from zero to four amino acids.
 2. A peptide according to claim 1 having the ability to bind and neutralize LPS which is the N-terminal region of a larger polypeptide.
 3. A peptide according to claim 1 having the ability to bind and neutralize LPS which is the C-terminal region of a larger polypeptide.
 4. A peptide according to claim 1 having the ability to bind and neutralize LPS which is inserted into the linear chain of a larger polypeptide.
 5. A peptide according to claim 1 wherein at least one amino acid of said sequence has been substituted by a non-natural homologous amino acid.
 6. A peptide according to claim 1 wherein the N-terminus has been modify by acetylation or succinylation.
 7. A polypeptide according to claim 2 wherein the N-terminus has been modify by acetylation or succinylation.
 8. A peptide according to anyone of claim 1 or 3 wherein the C-terminus is a —OH, —COOH or —CONH₂ group.
 9. A peptide according to claim 1 that has been constrained to adopt a cyclic conformation by an intramolecular disulfide or amide bond.
 10. A peptide according to claim 5 that has been constrained to adopt a cyclic conformation by an intramolecular disulfide or amide bond.
 11. A peptide according to claim 1 wherein the chain backbone have been substituted by backbone-mimetic organic entities.
 12. A peptide according to anyone of claim 5, 6, 9 or 10 wherein the chain backbone have been substituted by backbone-mimetic organic entities.
 13. A peptide according to anyone of claim 1, 5, 6, 9 or 10 wherein at least one amino acid of said sequences. have been substituted by alkylation using chemical or enzymatic methods.
 14. A peptide according to anyone of claim 1, 5, 6, 9 or 10 wherein at least one amino acid of the said sequences have been glycosylated using chemical or enzymatic methods.
 15. A linear polypeptide chain containing two or more repeats of a peptide sequence according to anyone of claim 1 or 5 connected by 12-25 amino acid linkers, rich in glycine, alanine, proline or serine residues.
 16. An arrangement of three of more copies of homologous peptide sequences or combinations of different sequences, according to anyone of claim 1 or 5, linked by their C-terminus to a lysine core structure.
 17. A pharmaceutical composition comprising effective amounts of a peptide according to claim 1, and a pharmaceutically acceptable diluent, carrier or adjuvant.
 18. A pharmaceutical composition comprising effective amounts of a molecule according to anyone of claims 2 to 4, and a pharmaceutically acceptable diluent, carrier or adjuvant.
 19. A pharmaceutical composition comprising effective amounts of a molecule according to claim 5, and a pharmaceutically acceptable diluent, carrier or adjuvant.
 20. A pharmaceutical composition comprising effective amounts of a molecule according to anyone of claims 6 to 16, and a pharmaceutically acceptable diluent, carrier or adjuvant.
 21. The use of the pharmaceutical composition according to claim 17 for the treatment of Systemic Inflammatory Response Syndrome.
 22. The use of the pharmaceutical composition according to claim 17 for the treatment of Gram-negative sepsis and its sequelae.
 23. The use of the pharmaceutical composition according to claim 17 for the treatment of obstructive jaundice.
 24. The use of the pharmaceutical composition according to claim 17 for the treatment of inflammatory bowel diseases.
 25. The use of the pharmaceutical composition according to claim 17 for the treatment of bacteremia.
 26. The use of the pharmaceutical composition according to claim 17 for the treatment of osteomyelitis.
 27. The use of the pharmaceutical composition according to claim 17 for the treatment of patients at risk of developing sepsis.
 28. The use of the pharmaceutical composition according to claim 17 for methods to treat chronic infections, arthritis or rheumatic disorders. 